4.1 Ethics statement: All work with infectious SARS-CoV-2 was performed in a containment level 3 laboratory at the Vaccine and Infectious Disease Organization according to approved protocols. The University of Saskatchewan’s University Animal Care Committee (UACC) and Animal Research Ethics Board (AREB) approved the animal work as per guidelines of the Canadian Council of Animal Care's (CCAC) criteria. Experimental protocols for mice studies were reviewed and approved by the canton of Vaud Veterinary Authority as per guidelines of the Swiss Federal Law on the Protection of Animals regulations.
4.2 Cells and viruses: All virus isolates/variants were isolated and/or propagated in Vero’76 cells (CRL-1587, ATCC) using previously established methods and authenticated by next-generation sequencing30,50. The ancestral SARS-CoV-2 Wuhan strain (GISAID Accession EPI_ISL_425177), Alpha (GenBank Accession# OQ781006), Beta (GenBank Accession# OM131550.1), Delta (GenBank Accession#: OM131551.1), and Omicron (GenBank Accession# OM131552.1)30 VOCs were isolated from Canadian patients and cultured on Vero’76 cells.
4.3 Structure prediction: The predicted three-dimensional coordinates of three constructs were produced and built through AlphaFold2 (AlphaFold2 (Colab-Fold) using MMseqs2 notebook and/or ChimeraX plugin for ALphaFold2/ Colab-Fold with default criteria) and validated using SWISS-MODEL structural assessments51,52. PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC) and ChimeraX were used to visualize the conformations. These constructs were submitted as a single protein. Next, we conducted energy minimization on generated models through ModRefiner, which employs physics- and knowledge-based force field in two steps to relax the structure. The process begins by constructing the main-chain structures using initial Ca atoms, followed by the refinement of sidechain rotamers in conjunction with the backbone atoms. The Ramachandran plot for three constructs show the residues are mapped in the allowed area for 93.8% for VIDO4372 and over 95% for both VIDO4304 and VIDO429653,54.
4.4 Expression and purification of VIDO4296, VIDO4304, and VIDO4372 chimeric protein and vaccine formulation: VIDO4296 chimeric protein was expressed by transient transfection and stable episomal maintenance in HEK 293 cells whereas VIDO4304, and VIDO4372 chimeric proteins were expressed in ExpiCHO cells as per manufacturer's instruction (ThermoFisher Scientific). In brief, cells were transfected with 0.8 µg/mL of plasmid DNA and subsequently cultured for 3-8 days until protein production reached peak levels or cell viability began to drop. The suspension culture containing the proteins was harvested by centrifugation at 4000 x g for 30 min at 4°C. After centrifugation, 300 mM NaCl and 0.05% Tween 80 was added to the clarified supernatant containing the expressed proteins. A final filtration through a 0.2 µm filter was performed and the clarified supernatant was 8-10x concentrated via tangential flow filtration using a 30kDA cut off membrane. The concentrated supernatant was diluted 1:1 in 2x PBS pH 8 right before loading onto a column with Repligen NGL Covid-19 Spike protein AR 2.0 resin. Following manufacturer’s recommendations, column volume and residence times were adjusted based on the expected protein in the sample. The columns were washed with 10 CV of PBS pH 7.5 before elution in 8 CV of 100 mM sodium acetate pH 5.0 + 1 M arginine. After elution, the protein was immediately buffer exchanged into a PBS buffer containing 20 mM sodium phosphate and 277 mM sodium chloride. The pH of the buffer was in the range of 6.5-7.5 which was chosen at least 0.5 unit below the theoretical pI of the protein, as predicted by Expasy ProtParam tool. The identity of the S1 chimeric proteins was confirmed by SDS-PAGE and immunoblotting. The concentration of the purified proteins was determined by BCA protein assay (Pierce®). The purified proteins were separated on a 4-20% gradient Mini-PROTEAN TGX gel (Biorad). For Western blot analysis, the proteins were transferred after SDS-PAGE onto 0.2 µm nitrocellulose using Trans Turbo Transfer pack (BioRad) according to the manufacturer’s recommendations. The membrane was blocked with 3% ECL prime blocking agent (Cytiva) in PBS buffer. Following standard blocking and washing procedure, the membrane was probed with in-house generated rabbit polyclonal primary antibody CoV19-S1 at 1:10,000 dilution. The immunoblot was visualized with a Li-Cor® NIR scanner using Li-COR® secondary anti-rabbit IRDye 800CW antibody at a dilution of 1:10,000. Sepivac SWE™ adjuvant (Seppic, France) was formulated with chimeric protein in a 1:1 ratio.
4.5 Animal vaccination and challenge: Six to eight weeks old male Syrian golden hamsters (Charles River Laboratories, Wilmington, USA) were vaccinated twice intramuscularly (IM) with either VIDO4296, VIDO4304, or VIDO4372 chimeric antigens formulated in SWE adjuvant. Three weeks after the second vaccination, animals were challenged intranasally (IN) with either the Beta, Delta, or Omicron SARS-CoV-2 VOC. Animal weight and clinical signs were monitored throughout the study. At 5-days post challenge (dpc), half of the animals from each group were euthanized and nasal washes and lung samples were collected for virological and pathological analysis. At 10 dpc, the remaining animals were euthanized as above. Sera were collected before each immunization, before the challenge, and at euthanasia.
4.6 Enzyme-linked immunosorbent assay (ELISA): SARS-CoV-2 S1-specific IgG was evaluated in serum samples by standard ELISA. Briefly, plates were coated with recombinant ancestral SARS-CoV-2 Wuhan S1 protein at a concentration of 1 µg/mL. Four-fold serially diluted serum samples were added to the coated plates. Antigen-specific antibodies were detected with Goat anti-Hamster IgG HRP at 1:7000 dilution (Thermo Fisher Scientific). Plates were developed with OPD peroxidase substrate (Thermo Fisher Scientific) and absorbance was read at 490 nm in a SPECTRAmax 340 PC Microplate Reader (Molecular Devices, CA, USA).
4.7 Virus neutralization assay: The serum samples were tested for viral neutralization (VN) against the SARS-CoV-2 Beta, Delta and Omicron VOCs. Serum samples were heat-inactivated at 56°C for 30 min and then serially diluted 1:2 in 96-well plates (Corning Incorporated, Corning, NY). Diluted samples were mixed with the virus at a 1:1 ratio for 1 h at 37°C. The virus-serum sample mixtures were transferred to Vero’76 cell monolayers and incubated at 37°C. Endpoint neutralization titer was based on 100% inhibition of cytopathic effect (CPE) observed on day 5 after cell infection.
4.8 Infectious virus titration assay: Infectious virus titration assays were carried out on nasal washes and lung samples. Nasal washes were collected both before and after the challenge. At necropsy, lung samples were collected, weighed, and homogenized in serum-free DMEM using a Qiagen TissueLyzer. After adding serially diluted samples to Vero’76 cells for an hour, the samples were removed and replaced with fresh DMEM medium containing 2% FBS. Cells were examined for CPE five days after infection, and the 50% endpoint (TCID50) was determined using the Spearman–Kärber method 55,56.
4.9 Stability and immunogenicity testing of a “one-vial” vaccine formulation: The adjuvant Sepivac SWE™ is a squalene-based oil-in-water adjuvant, comprising squalene (3.9%, w/v), sorbitan trioleate (0.47%, w/v), and polyoxyethylene (80) sorbitan monooleate (0.47%, w/v) dispersed in 10 mM citrate buffer at pH 6.5. Sepivac SWE™ was developed by the Vaccine Formulation Institute (Switzerland) and made commercially available via an open access model by Seppic (France). VIDO4296, VIDO4304, and VIDO4372 chimeric proteins were formulated with SWE adjuvant in a 1:1 ratio and formulations were made isotonic by addition of sodium chloride. The pH of the VIDO4296 + SWE and VIDO4372 + SWE formulations were adjusted to pH 6.5, while VIDO4304 + SWE was adjusted to pH 6.7.
Formulations were vialed in ISO 2R type 1 glass vials (Schott) using bromobutyl 13 mm stoppers (West) and 13 mm flip-off aluminum seals (West) and stored at 2-8°C for stability studies up to 13 months. All quality control assays were performed in triplicate at specified time points for stability evaluation. Formulations were characterized by assessing visual appearance, pH (SevenCompact, Mettler Toledo, Switzerland), particle size, polydispersity index and zeta potential by dynamic/electrophoretic light scattering (Zetasizer Nano ZS, Malvern Panalytical, UK), osmolality (OsmoPro, Advanced Instruments, USA), squalene concentration by reverse-phase high pressure liquid chromatography (1260 Infinity II, Agilent, USA), and antigen integrity by biolayer interferometry (Octet Red 96e, Sartorius, USA). In vivo immunogenicity studies were carried out at specified time points to confirm potency.
BLI assays were performed using the Octet Red96e instrument (Sartorius, USA). Protein A biosensors were dipped for 60 s into wells containing running buffer (PBS pH 7.2 with 0.1% w/v bovine serum albumin and 0.02% v/v tween 20) for baseline measurement. CR3022 recombinant Anti-SARS-CoV-2 Spike Glycoprotein S1 antibody (Abcam) was loaded at a concentration of 10 µg/mL onto biosensors for 300 s followed by a 40 s second baseline measurement in running buffer. VIDO4296 + SWE, VIDO4304 + SWE, and VIDO4372 + SWE formulations were diluted from 1:4 to 1:16 in running buffer. The sensors were dipped into wells containing diluted formulations for 200 s to assess association. Curves were aligned at the association step and antigen concentration was determined using a 4P-unweighted dose response analysis based on initial slope of binding rates.
Six weeks old female BALB/c mice (Charles River Laboratories, France) were immunized intramuscularly (IM) at day 0 and day 28 with extemporaneous formulations of either VIDO4296, VIDO4304, or VIDO4372 chimeric antigens adjuvanted with Sepivac SWE, or preformulated preparations stored at 2-8°C in stability studies. The control group of mice was immunized with PBS without any adjuvant. At day 42, mice were euthanized, and sera were collected for antibody response analysis.
SARS-CoV-2 S1-specific total IgG was evaluated in serum samples by standard ELISA. Briefly, plates were coated with soluble SARS-CoV-2 RBD at a concentration of 1.25 µg/mL. Four-fold serially diluted serum samples were added to the coated plates. Antigen-specific antibodies were detected with Goat anti-Mouse IgG(H+L) HRP (Southern Biotech). Plates were developed with OPD peroxidase substrate (ThermoFisher Scientific) and absorbance was read at 492 nm in a Synergy H1 Microplate Reader (Biotek, USA).
The serum samples were tested for viral neutralization (VN) using the SARS-CoV-2 surrogate virus neutralization test (sVNT) kit (Genscript) against different VOCs. The sVNT assay uses purified RBD from the SARS-CoV-2 S protein and host cell receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). This highly specific interaction can then be inhibited, i.e., blocked by the neutralizing antibodies (Nabs) in mice sera in the same manner as in a conventional VNT. Serum samples were diluted from 1:10 to 1:200 in 96-well plates (Corning Incorporated, Corning, NY). Diluted samples were mixed with HRP labeled SARS-CoV-2 RBD proteins (RBD-HRP), wild type, Delta-, Omicron-, or Beta-VOC (Genscript), at a 1:1 ratio for 30 min at 37°C. The RBD-HRP-serum sample mixtures were transferred to a ACE2-coated capture plate and incubated for 15 min at 37°C. Plates were developed with TMB substrate (Genscript) and absorbance was read at 450 nm in a Synergy H1 Microplate Reader (Biotek).
4.10 Statistical analysis: Graphs and descriptive statistical analyses were performed on GraphPad Prism version 9. Statistical comparisons among different groups were conducted using either Two-Way ANOVA followed by Sidak’s method for multiple comparison or One-Way ANOVA followed by Kruskal-Wallis test.